Genome sequence of the chromate-resistant bacterium Leucobacter salsicius type strain M1-8^T

16S rRNA analysis

A representative genomic 16S rRNA gene of strain M1-8^T was compared with those obtained using NCBI BLAST [17] with the default settings (only highly similar sequences). The most frequently occurring genera were Leucobacter (65.0%), unidentified bacteria (20.0%), Curtobacterium (6.0%), Microbacterium (5.0%), Leifsonia (2.0%), Subtercola (1.0%), and Zimmermannella (1.0%) (100 hits in total). The species with the Max score was Leucobacter exalbidus (AB514037), which had a shared identity of 99.0%.

The multiple sequence alignment program CLUSTALW [18] was used to align the 16S rRNA gene sequences from M1-8^T and related taxa. Phylogenetic trees were constructed based on the aligned gene sequences using the maximum-likelihood, maximum-parsimony, and neighbor-joining methods based on 1,000 randomly selected bootstrap replicates using MEGA version 5 [19]. Strain M1-8^T shared 99.1% nucleotide sequence similarity with L. aerolatus Sj10^T, the closest validated Leucobacter species according to the phylogeny (Figure 1). Figure 1 shows the phylogenetic position of L. salsicius in the 16S rRNA-based tree. The sequence of the single 16S rRNA gene copy found in the genome did not differ from the previously published 16S rRNA sequence (GQ352403).

Morphology and physiology

Strain M1-8^T is classified as class Actinobacteria, order Actinomycetales, family Microbacteriaceae, genus Leucobacter (Table 1) [1]. The strain L. salsicius M1-8^T was isolated from a Korean salt-fermented food that contains tiny shrimp (shrimp jeotgal). The cells of strain M1-8^T were rod-shaped, 1.0–1.5 µm in length, and 0.4–0.5 µm in diameter (Figure 2). No flagella were observed. The colonies were cream in color and circular with entire margins on marine agar medium. Strain M1-8^T was aerobic and Gram-positive (Table 1). Optimum growth was observed at 25–30°C, at pH 7.0–8.0, and in the presence of 0–4% (w/v) NaCl. The tolerance of Cr (VI) was observed at up to 10.0 mM K₂CrO₄. The physiological characteristics, such as the growth substrates of M1-8^T, were described in detail in a previous study [1].

MIGS ID	Property	Term	Evidence codea
	Current classification	Domain Bacteria	TAS [24]
		Phylum Actinobacteria	TAS [25]
		Class Actinobacteria	TAS [26]
		Order Actinomycetales	TAS [26–29]
		Family Microbacteriaceae	TAS [26,27,30,31]
		Genus Leucobacter	TAS [2]
		Species Leucobacter salsicius	TAS [1]
		Type strain M1-8T	TAS [1]
	Gram stain	Positive	TAS [1]
	Cell shape	Rod-shaped	TAS [1]
	Motility	Non-motile	TAS [1]
	Sporulation	Not reported
	Temperature range	Mesophile	TAS [1]
	Optimum temperature	25–30°C	TAS [1]
	pH	pH 7–8	TAS [1]
MIGS-22	Oxygen requirement	Aerobic	TAS [1]
	Carbon source	Heterotroph	TAS [1]
	Energy metabolism	Not reported
MIGS-6	Habitat	Fermented food	TAS [1]
MIGS-6.3	Salinity	Halotolerant	TAS [1]
MIGS-15	Biotic relationship	Free-living	NAS
MIGS-14	Pathogenicity	Not reported	NAS
	Isolation	Fermented food (Shrimp jeotgal, a Korean salt-fermented food)	TAS [1]
MIGS-4	Geographic location	South Korea	TAS [1]
MIGS-5	Sample collection date	May 2009	NAS
MIGS-4.1	Latitude	Not reported
MIGS-4.1	Longitude	Not reported
MIGS-4.3	Depth	Not reported
MIGS-4.4	Altitude	Not reported

The evidence codes are as follows. TAS: traceable author statement (i.e., a direct report exists in the literature). NAS: non-traceable author statement (i.e., not observed directly in a living, isolated sample, but based on a generally accepted property of the species, or anecdotal evidence). These evidence codes are derived from the Gene Ontology project [32].

Figure 2 Scanning electron micrograph of Leucobacter salsicius M1-8^T, which was obtained using a SUPRA VP55 (Carl Zeiss) at an operating voltage of 15 kV. The scale bar represents 1 µm.

Chemotaxonomy

The peptidoglycan hydrolysate from strain M1-8^T contained alanine, 2,4-diaminobutyric acid (DAB), γ-aminobutyric acid (GABA), glutamic acid, and glycine. The predominant fatty acids (>10% of the total) in M1-8^T were anteiso-C_15:0 (63.6%), anteiso-C_17:0 (16.7%), and iso-C_16:0 (14.2%). The polar lipid profile of strain M1-8T contained diphosphatidylglycerol and an unknown glycolipid. The major menaquinone in M1-8^T was MK-11 and the minor menaquinones were MK-10 and MK-7.

Genome project history

Growth conditions and DNA isolation

L. salsicius strain M1-8^T was cultured aerobically in marine agar medium at 30°C. Genomic DNA was extracted using a G-spin DNA extraction kit (iNtRON Biotechnology), according to the standard protocol recommended by the manufacturer.

Genome sequencing and assembly

The genome was sequenced using a combination of an Illumina Hiseq system with a 150 base pair (bp) paired-end library, a 454 Genome Sequencer FLX Titanium system (Roche) with an 8 kb paired-end library, and a PacBio RS system (Pacific Biosciences). The Illumina reads were assembled using CLC Genomics Workbench ver. 5.0. The initial assembly was converted for the CLC Genomics Workbench by constructing fake reads from the consensus to collect the read pairs in the Illumina paired-end library. The 454 paired-end reads were assembled with Illumina data using gsAssembler ver. 2.6 (Roche) and the PacBio sequences were clustered into overlapping assembled data. CodonCode Aligner and CLC Genomics Workbench 5.0 were used for sequence assembly and quality assessment in the subsequent finishing process. The Illumina (189.78-fold coverage; 4,003,590 reads), PacBio (88-fold coverage; 23,441 reads), and 454 sequencing (7.96-fold coverage; 130,181 reads) platforms provided 213.62 × coverage (total 4,157,212 sequencing reads) of the genome. The final assembly identified one scaffold that included 28 contigs.

Genome annotation

The genes in the assembled genome were predicted using Integrated Microbial Genomes - Expert Review (IMG-ER) platform as part of the DOE-JGI genome annotation pipeline [35], followed by a round of manual curation using the JGI GenePRIMP pipeline. Comparisons of the predicted ORFs using the SEED [36], NCBI COG [37], Ez-Taxon-e [38], and Pfam [39] databases were conducted during gene annotation. Additional gene prediction analyses and functional annotation were performed with the Rapid Annotation using Subsystem Technology (RAST) server databases [40] and the gene-caller GLIMMER 3.02. RNAmer 1.2 [41] and tRNAscan-SE 1.23 [42] were used to identify rRNA genes and tRNA genes, respectively. The CLgenomics^TM 1.06 (ChunLab) was used to visualize the genomic features.